Dorothy E. Schumm

Transport of informosomes from isolated nuclei of regenerating rat liver

Abstract Nuclei, isolated from regenerating rat liver following a 30-minute prelabelling in vivo with orotic acid-6- 14 C, are shown to release rapidly labelled RNA in informosome-like particles in the cell-free system. This release is both energy- and temperature-dependent and is reduced if the cytosol in the incubation medium is derived from normal, rather than regenerating liver. The identity of the transported RNA with messenger RNA is supported by its failure to compete with ribosomal, or transfer RNA under optimal hybridizing conditions with rat liver DNA, and also by its poly-A content and occurrence in nucleo-protein particles with densities characteristic of informosomes.

Insulin-modulated transport of RNA from isolated liver nuclei

Abstract The addition of 3 × 10 −7 m insulin to a cell-free RNA transport system caused an increase of 50% in the release of messenger-like RNA from 30-min prelabeled rat liver nuclei. Insulin concentrations above 1.2 × 10 −6 m inhibited RNA release. These hormonal effects were not observed when nuclei were prepared from the insulin-resistant Zucker rat (fa/fa), while the level of stimulation in the heterozygote was approximately one-half that observed with normal liver nuclei. Nuclei prelabeled for 120 min and releasing predominantly ribosomal RNA also did not respond to insulin added to the cell-free system. The hormone appears to affect primarily mRNA transport rather than processing.

Early biochemical changes in phytohemagglutinin-stimulated peripheral blood lymphocytes from normal and tumor-bearing rats

Abstract The very early increase in the intracellular concentration of 3′,5′-cyclic GMP and in the rate of incorporation of 33 P into membrane phospholipid, and the early increases in the level of uridine kinase and cystathionine synthetase upon stimulation of peripheral lymphocytes with phytohemagglutinin, correlate well with the standard assay for lymphoblastic transformation. The rapidity, lower variability and in particular greater sensitivity of assays based on earlier events, suggests that they may be useful adjuncts for assessing immunocompetence under specified conditions, and in combination, may aid in diagnosing the molecular lesion when immunity is impaired. Coincidentally this study also shows that the peripheral blood lymphocytes from animals bearing transplantable tumors are inherently incompetent, and also that the serum from such animals suppresses the transformation of lymphocytes from normal animals. The immunosuppressive effect of the transplantable tumors was independent of the adrenal cortex.

The in vivo equivalence of a cell-free system for RNA processing and transport

Abstract The equivalence of messenger RNA released (transported) from isolated rat liver nuclei to three selected media, with messenger RNA normally released to liver cytoplasm in vivo , has been evaluated by competitive DNA: RNA hybridization. Near normal nuclear restriction was exhibited by nuclei in media fortified with ATP, salts, spermidine and dialyzed cytosol. The RNA transport in the latter system was markedly inhibited by colchicine as was also the transport of RNA in vivo . Both nuclear restriction and sensitivity of the RNA transport to colchicine in media lacking spermidine and cytosol deviated significantly from the in vivo norm. The results emphasize the importance of establishing the in vivo equivalence in cell-free systems designed to study RNA synthesis, processing and transport.

Age-dependence of nuclear RNA processing.

Following a 3 hour in vivo labelling of cytoplasmic RNA in rat liver with orotic acid-6-14C under conditions where ribosomal RNA synthesis was suppressed, the proportion of labelled messenger-like RNA released to the cytoplasm which contained polyadenylate (poly(A)) tracts was about 3.0 times higher in the livers of juvenile (50 day) as compared to adult (180 day) rats. This discrepancy was confirmed in a cell-free system which consisted of isolated prelabelled nuclei in fortified cytosol. Thus under conditions where approximately 80% of the released labelled RNA was messenger-like, the proportion of polyadenylated labelled RNA transported to the homologous cytosol was 3.4-fold greater in the systems derived from juvenile as compared to adult rat liver. Through comparisons of homologous and heterologous systems it was determined that the age-dependent change in the metabolism of polyadenylated messenger RNA resides in the nucleus and not in the cytoplasm. This change, furthermore does not involve the known ATP-dependence of nuclear RNA release. Rather it must involve other age-dependent changes in the processing or transport of polyadenylated messenger RNA.

Differential effect of ATP on RNA and DNA release from nuclei of normal and neoplastic liver

Abstract Nuclei isolated from differentiated normal liver, moderately differentiated Hepatoma 5123 and poorly differentiated Novikoff hepatoma have been compared with respect to ATP-dependence of messenger-like RNA release and resistance to lysis (DNA release) in a cell-free system containing homologous cytosol. The release of RNA from the nuclei of liver, Hepatoma 5123D and the Novikoff hepatoma was totally ATP-dependent, partially ATP-dependent and ATP-independent, repectively. The sensitivity of the nuclear RNA transport in the presence of ATP to beryllium nitrate, an inhibitor of a nuclear pore phosphatase, paralleled their ATP-dependence. Although RNA release from the nuclei of both liver and Novikoff hepatoma has an absolute requirement for cytosol proteins, the structural integrity of liver, but not Novikoff hepatoma nuclei in the presence of ATP, is dependent on macromolecules in the cytosol.

Changes in nuclear RNA transport incident to carcinogenesis

Abstract Rats were treated with the hepatocarcinogens thioacetamide and dimethylnitrosamine and the release (transport) of RNA from the isolated liver nuclei to homologous or heterologous liver cytosol was evaluated in a cell-free system at various times after treatment. Within 24 hr of treatment, cytosol from the carcinogen-treated animals enhanced the release of RNA from liver nuclei of untreated rats. This enhanced transport capacity of the cytosol persisted up to 4 months after treatment; furthermore, the RNA transport from the liver nuclei of carcinogen-treated animals showed a partial loss of its ATP-dependence. Although the capacity of cytosol from treated animals to support RNA transport dropped below control levels by 9 months after treatment, the RNA transport from nuclei of the carcinogen-treated animals remained partially ATP-independent. This ATP-independence, which is also a characteristic of nuclei from hepatomas, is not a characteristic of the age of the animal, nor is it due to differences in the pool size of nuclear ATP or the requirement for polyadenylation of messenger RNA for transport.

Differential effect of plasma fractions from normal and tumour-bearing rats on nuclear RNA restriction

WE have already presented preliminary evidence1 that crude plasma from tumour-bearing rats contains elevated concentrations of macromolecules which stimulate the release of messenger RNA (mRNA) from isolated nuclei. The detection of these putative regulatory components was facilitated by a cell-free system developed originally to study messenger2–5 and ribosomal6,7 RNA (rRNA) processing and transport in vitro. Since both maximal RNA transport and normal nuclear RNA restriction in vitro (that is, equivalence between messengers released from nuclei in vivo and in vitro) require the presence of macromolecules from homologous cytosol2,8 it is possible that the tumour cell cytoplasm is the source of the putative regulatory macromolecules in the peripheral blood of tumour-bearing animals. This possibility is supported by evidence9,10 that factors involved in the regulation of cellular proliferation are released to circulation from tumour cells, and by the observation11 that the enzyme composition of the host liver of tumour-bearing rats converges to that of the tumour. The spectrum of messengers transported from normal rat liver nuclei in homologous cytosol is significantly different from that transported in hepatoma cytosol3, suggesting that differences exist in these putative regulatory components of the two tissues.

Transport of functional messenger RNA from liver nuclei in a reconstituted cell-free system

The reliability of a reconstituted cell-free system for messenger RNA processing and transport, consisting of isolated nuclei in fortified cytosol, has been evaluated in terms of the functionality and regulated release of the transported product. The poly(A) messenger RNA transport in vitro formed appropriate initiation complexes with ribosomes in an optimized translation system and had template activity comparable to that transported in vivo. The intra-nuclear origin of this messenger RNA is supported by pulse-labeling studies, its transport from detergent-treated nuclei and the absence of the release under non-transport conditions. Serum albumin was identified by immunoprecipitation and electrophoresis as one of the products synthesized when the transported RNA was translated in vitro. The transport of messenger RNA in the cell-free system was dependent on specific cytosol (soluble cytoplasmic) proteins. These proteins, which constitutes less than 0.1% of the total cytosol proteins, are precipitated wtih streptomycin with high specificity.

An oncofetal 60-kilodalton protein in the plasma of tumor-bearing and carcinogen-treated rats

A Mr 60,000 protein, detected by its ability to induce the release of RNA from isolated nuclei, is present in the plasma of tumor-bearing and carcinogen-treated rats, together with low amounts of 2 messenger RNA transport proteins identified earlier in normal cells. The Mr 60,000 protein has been identified in tumor cell cytoplasm and in amniotic fluid, but does not appear to cross the placental barrier. Significant amounts of the Mr 60,000 oncofetal protein appear in the plasma of carcinogen-treated rats within a few weeks of treatment. It may be the fetal form of the adult messenger RNA transport proteins.